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My question is related to protein purification using a ÄKTA FPLC. We used StrepTrap HP Columns (1 ml column Volume (CV)) from GE Healthcare Life Sciences to purify a strep-tagged protein. In the first approach, this worked pretty fine. We wanted to re-use the columns after regenerating them according to the official protocol. The included steps were all performed at a flow rate of 1 ml/min and with filtered solutions:

  1. 3 CV distilled water
  2. 3 CV 0.5 M NaOH
  3. 3 CV distilled water
  4. 5 CV 20 % EtOH
  5. Storage of columns in 20 % EtOH at 4°C.
  6. Equilibration of the columns with Binding Buffer just before next use.

By adhering to the protocol we encountered some problems with the next purification (it was the same protein, but a new sample of cell extract). The purification worked less effective and the amount of purified protein was very low.

We tried a slightly different protocol for regeneration, also at a flow rate of 1 ml/min and filtered solutions.

  1. 5 CV distilled water
  2. 5 CV 20 % EtOH
  3. 3 CV 6 M Guanidinhydrochloride (sol.)
  4. 5 CV 0.5 M NaOH
  5. 3 CV distilled water
  6. 5 CV 20 % EtOH
  7. Storage in 20 % EtOH at 4°C
  8. Equilibration with Binding Buffer before next use.

We chose the Guanidinhydrochloride to denaturate all remaining proteins from the column resin and support the regeneration by this. But also with this protocol we encountered the same problems (low efficiency of purification).

I would be very interested if anyone of you have encountered the same problems and maybe have a solution/better protocol for this regeneration step.

Thank you for your suggests in advance! :)

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  • $\begingroup$ What was the exact problem with the second purification (eg poor binding, poor elution, etc). Did anything change with the preparation of the second sample? Did you use desthiobiotin for the first elution? Is there a lot of biotin in the culture medium? $\endgroup$
    – canadianer
    Commented Jan 3, 2020 at 11:17
  • $\begingroup$ We used desthiobiotin (2.5 mM in Binding Buffer (Tris, NaCl, EDTA)) as Elution buffer. The second sample was prepared exactly the same way as the one before (same incubation time, same induction of protein expression). Additionaly we tried it with another protein (with a new column) and observed the same problem. Meaning: First purification was rather good, the second was not so nice. The culture medium contained 0.2 g/l Biotin (CGXII+Glucose). What we observed during the FPLC was, that the peak for the eluted protein was not very high. We are not sure, if this is because of poor binding. $\endgroup$
    – Florian
    Commented Jan 3, 2020 at 11:29
  • $\begingroup$ Or because of poor elution. We tested the flowthrough on an SDS-PAGE and it seems in both cases (the first attempt, where the purification was fine, and the one where it was not) that we have enough protein in the sample (strong bands at the expected MW of our protein in the cell extract and still in the flowthrough.). $\endgroup$
    – Florian
    Commented Jan 3, 2020 at 11:32
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    $\begingroup$ How much of your target protein is in the flow through compared to the sample that was applied? That should tell you how well it is binding. You might want to think about how much of the biotin in the culture medium is making it to your column. Biotin will bind with very high affinity and prevent binding of the strep-tagged protein. It might be irrelevant but is perhaps worth considering. $\endgroup$
    – canadianer
    Commented Jan 3, 2020 at 12:12
  • $\begingroup$ Is there a technique to determine the amount of target protein in the cell extract and in the flow through? I guess a BCA Assay e.g. would just give you the amount of total protein in the sample? The remaining Biotin is a very good point I have not thought about at this point! Is a HABA assay an appropriate way to measure the Biotin amount in the sample? $\endgroup$
    – Florian
    Commented Jan 3, 2020 at 12:30

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